MicroCT Advanced Imaging of the Foot and Ankle: Technique Guide

Abstract

Background:

Recent advances in micro-computed tomography (MicroCT) imaging have enabled detailed investigations of human microvascular anatomy, providing new insights that may influence treatment options and optimize local reparative potential. This article describes a reproducible cadaveric perfusion technique for visualizing foot and ankle microvasculature using MicroCT, designed to support anatomical research and surgical planning studies.

Methods:

Ten matched pairs of fresh-frozen cadaveric lower limbs were used to develop this protocol. An 18-gauge angiocatheter was used to cannulate the anterior and posterior tibial arteries for perfusion of the foot and ankle, or the popliteal artery for perfusion of the entire lower leg. Clearing was performed sequentially with 0.9% saline, 3% hydrogen peroxide, and water. Perfusion was performed with a 50% barium sulfate/2.5% gelatin solution. Confirmatory images were obtained using mini c-arm fluoroscopy. Final images were obtained for microvascular assessment using a commercial MicroCT scanner. Integrity of the perfusate was visually evaluated on MicroCT over the course of 4 freeze-thaw cycles spanning 2 months.

Results:

All intraosseous and extraosseous microvascular structures were successfully visualized using MicroCT of the cadaveric lower extremities. Microvasculature was perfused in continuity without incidence of contrast extravasation. When present, intraosseous nutrient arteries of the first and fifth metatarsal, and branches of the tarsal sinus artery were appreciated. Contrast material remained visually consistent even after preforming surgical resections and undergoing multiple freeze-thaw cycles.

Conclusion:

This standardized perfusion technique was effective in the visualization of microvasculature in the foot and ankle. In addition to 3-dimensional mapping using MicroCT, this reproducible protocol can be used in numerous advanced imaging applications, including microvascular assessment following surgical reconstructions and instrumentation.

Clinical Relevance:

A refined understanding of the microvascular anatomy of the foot and ankle using MicroCT perfusion imaging can potentially guide surgical techniques to minimize iatrogenic injury and optimize healing.

Visual Abstract.

This is a visual representation of the abstract.

Introduction

There are various imaging modalities to visualize and assess the vasculature of extremities. These range from anatomic and radiologic tests such as color duplex ultrasound (CDUS) to advanced imaging techniques including computed tomography angiography (CTA) and magnetic resonance angiography (MRA). These modalities have demonstrated utility in the in vivo evaluation of distal blood flow, particularly in pathologies such as peripheral arterial disease (PAD).^3,5

The Spalteholz technique was first introduced in 1914, ⁸ as a solvent-based method to produce transparent 3-dimensional specimens for the evaluation of vascular and osseous anatomy, and has since undergone many modifications.^6,9,11 Although these plastination techniques have provided insight into gross extraosseous and intraosseous anatomy, they are often cumbersome and time-intensive. Furthermore, they are generally limited in assessing fine microvascular anatomy (arterioles) and osseous architecture (trabecular anatomy), which require a much higher resolution only achieved through advanced micro-computed tomography (MicroCT). For visualizing arterioles and trabecular anatomy, resolution with voxel sizes of less than 30 μm is ideal, although finer resolution can be achieved at the expense of longer scan times.

Emphasis on microvasculature has several key implications, including the evaluation of local reparative potential,^4,7,10 identifying conduits for reconstructive procedures such as digital transplants, ¹ and mapping relative “safe zones” for performing bony osteotomies. ² Improved understanding of the complex angioarchitecture of the foot and ankle may refine our understanding of why certain joints are more prone to nonunion and/or avascular necrosis, and ultimately may guide treatment options.

To visualize these structures effectively, a radiopaque contrast solution is usually infused before imaging. Previous cadaveric perfusion-based MicroCT studies have used a variety of contrast solutions.^2,4 However, to date, a standardized perfusion protocol for MicroCT advanced imaging has not yet been fully described. This work is intended as a technique guide to support future anatomical research and surgical planning studies using cadaveric models.

Methods and Materials

Perfusion Protocol

All perfusions were performed on 10 matched pairs of thawed fresh-frozen cadaveric lower limbs at room temperature (Table 1). For perfusion of the foot and ankle, the anterior and posterior tibial arteries were dissected out 5-10 cm proximal to the tibiotalar joint (Figure 1). This level was found to be adequate for perfusing all vascular anatomy of the foot and ankle. Alternatively, the popliteal artery was dissected out proximal to the anterior tibial artery branch to perfuse the entire lower leg. Once identified, each artery was clamped with a hemostat and an 18-gauge angiocatheter was introduced just distal to the clamp (Figure 2).

Table 1.

MicroCT Perfusion Imaging Technique.

Cannulation	18 g angiocatheter (anterior + posterior tibial arteries; popliteal artery)
Flushing	500 mL normal saline 500 mL 3% hydrogen peroxide 500 mL water
Perfusion	50% barium sulfate/2.5% gelatin
Confirmation	Confirm distal perfusion on fluoroscopy
Freeze	−4°C overnight for gelatin to solidify
Imaging	Resect areas of interest, scan using MicroCT a

^aA voxel size less than 30 μm is recommended to clearly visualize structures such as arterioles and larger.

Figure 1.

Perfusion of the foot and ankle can be accomplished by dissecting out and cannulating the anterior and posterior tibial arteries about 5-10 cm proximal to the tibiotalar joint (A). Alternatively, perfusion of the entire lower leg can be achieved by dissecting out and cannulating the popliteal artery just proximal to its branching points (B).

Figure 2.

A hemostat was used to clamp the artery ([A] posterior tibial artery, [B] popliteal artery) before introducing an 18-gauge angiocatheter just distal to the clamp.

For each extremity, a total of 500 mL of 0.9% saline was first introduced manually with gentle, constant pressure using a syringe (approximately 100 mm Hg). This was then followed by 500 mL of 3% hydrogen peroxide to remove any clots. Finally, a total of 500 mL of water was introduced in a similar fashion to remove the hydrogen peroxide. Adequate flushing of the vasculature was achieved when clear effluent was observed leaving the venous system.

A perfusate of 50% barium sulfate/2.5% porcine gelatin was created by adding 30 g of barium sulfate and 1.8 g of porcine gelatin to 60 mL of water, mixed with a tabletop mixer (Figure 3). If cannulation was performed via the anterior and posterior tibial arteries, 30 mL of perfusate was introduced into each vessel for a total of 60 mL. If cannulation was performed through the popliteal artery, the total volume of perfusate was doubled and introduced until resistance was encountered.

Figure 3.

A perfusate of 50% barium sulfate and 2.5% porcine gelatin was created by adding 30 g of barium sulfate and 1.8 g of porcine gelatin to 60 mL of water, mixed with a tabletop mixer.

Following perfusion, mini c-arm fluoroscopy was performed to confirm adequate distal vascular filling of the distal phalanges and calcaneus prior to MicroCT analysis (Figure 4). After confirmation, the angiocatheters were removed and the clamps left in place as the specimens were stored in a −4 °C freezer to allow the gelatin to solidify overnight.

Figure 4.

Fluoroscopy images confirming adequate vascular perfusion with the radio-opaque perfusate, as evidenced by clear visualization of vasculature in the (A) forefoot and (B) hindfoot.

MicroCT Imaging Protocol

Each resected specimen was carefully wrapped in gauze and secured in a radiolucent container to minimize micromotion during imaging. MicroCT imaging was performed using the Sky-Scan-1275 MicroCT (Bruker MicroCT, Billerica, MA; output 80 kV, current 125 μm) unit, although this can be reproduced with other similar imaging systems. For the metatarsals, images were acquired using an aluminum 1.0-mm filter with a voxel size of 27 μm in high resolution. The rotation step was set to 0.6 degrees with a frame rate average of 5. For more dense bone such as the talus, a Copper 1.0-mm filter was used instead, with all other parameters constant.

All images were qualitatively assessed for clarity and continuity of perfused microvasculature. Presence and location of nutrient vessels in the first and fifth metatarsals were documented, and named metatarsal and talar arteries were identified.

Multiple Freeze-Thaw Cycles

Quality of perfusion imaging was also evaluated over the course of multiple freeze-thaw cycles. Following initial scan, specimens were returned to a −20 °C freezer, where they remained for 2 weeks before being thawed in room temperature and rescanned. This process was repeated 4 times over the course of 8 weeks.

Results

MicroCT imaging analysis demonstrated full penetration of both the extraosseous and intraosseous vascular networks of the foot and ankle (Figure 5). Furthermore, no contrast extravasation was appreciated using this technique.

Figure 5.

Three-dimensional reconstructed images using MicroCT imaging software demonstrating (A) extraosseous dorsal metatarsal artery of the fifth metatarsal; (B) intraosseous branches of the tarsal sinus artery within the body of the talus; and (C) extraosseous vasculature of the first metatarsal including the first proximal perforating artery, deep plantar arterial arc, and first dorsal and plantar metatarsal arteries.

A predominant nutrient artery was identified originating from the deep plantar arterial arc and entering plantarmedially at the proximal one-third of the first metatarsal. In comparison, nutrient arteries were variable in number and location in the fifth metatarsal, with variability within matched pairs. The tarsal sinus artery was visualized giving off multiple intraosseous branches within the talar body once entering the tarsal sinus (Figure 5).

Consistent qualitative visualization was observed across repeated imaging sessions, including both initial and subsequent scans. Specimens underwent 4 freeze-thaw cycles over 8 weeks without compromising the quality and visualization of microvascular structures. Furthermore, en bloc resection following specimen perfusion did not lead to contrast extravasation or compromise image quality.

Pearls for Adequate Perfusion

There were several clinical signs that portended to an adequately perfused limb:

• Evidence of fluid extravasating through the venous system and through collateral arteries was indicative of adequate vessel penetration (ie, hydrogen peroxide bubbles and perfusate exiting the anterior tibial artery cannula when the posterior tibial artery was being flushed and perfused).

• Visible swelling of the foot after introduction of 3% hydrogen peroxide, with palpable crepitus throughout the foot due to air bubbles.

• Gross confirmation of adequate perfusion through direct visualization of vasculature on mini c-arm fluoroscopy.

Discussion

MicroCT imaging has emerged as a valuable tool for exploring the intraosseous and extraosseous anatomy of the distal extremities with substantial potential for the foot and ankle. These cadaveric studies employ various perfusion techniques, each with potential advantages and drawbacks. The results of the current study present a standardized perfusion protocol that allows for consistent visualization of the microvasculature of the foot and ankle.

MicroCT perfusion imaging has recently been employed using a variety of techniques to study vascular blood flow of the foot and ankle. For example, Finney et al ⁴ used a similar perfusion technique using 15% barium sulfate solution to visualize microvasculature of the lesser metatarsal plantar plate. The authors determined that there is increased vascular density in the distal and proximal poles of the plantar plate, with a relatively hypovascular midsubstance. In a follow-up study by the same group, Singer and colleagues used the same perfusion technique with MicroCT analysis to identify evidence of neovascularization in torn plantar plates. ⁷ Other perfusates have been described for MicroCT perfusion imaging, including a low-viscosity radiopaque polymer to visualize the microvascular supply to the first metatarsal head. ² The authors identified a relative safe zone about 25 mm proximal to the first metatarsal head for which osteotomies could be performed with low risk of arterial injury.

Although there are several viable options when choosing a perfusate, a solution of 50% barium sulfate with 2.5% gelatin is recommended because it is relatively inexpensive and easy to reproduce. Furthermore, it affords a longer working time as opposed to other perfusates that rely on rapid polymerization for the perfusate to solidify. ² With the addition of 2.5% gelatin, the perfusate is allowed to solidify in the refrigerator overnight. Notably, it was observed that subsequent sharp dissection and en bloc resection did not lead to contrast extravasation and/or compromise quality of MicroCT imaging. This is particularly important when perfusion imaging is performed to assess for iatrogenic microvascular injury.

Several modifications to the perfusion technique were trialed before arriving at the final perfusion protocol. First, determining the optimal pressure that should be applied, either manually or with a syringe pump, during the flushing and perfusion steps was critical. Regardless of the method, it is important to apply a constant physiologic pressure (100 mm Hg) when introducing solution, and to stop once encountering resistance to avoid vessel damage and subsequent contrast extravasation. Second, the 3% hydrogen peroxide solution would occasionally crystalize within the vessels, leading to lack of intravasation of the perfusate distally and poor image quality. The additional step of clearing the hydrogen peroxide with 500 mL of water was found to be effective in allowing the contrast solution to perfuse unhindered.

Limitations to this study include lack of objective and quantitative metrics, as all findings were recorded qualitatively. Specifically, future studies should incorporate quantitative measurements such as vessel density within the talar neck and base of the fifth metatarsal, 2 classic “watershed” areas. In addition, portions of the perfusion technique were based on subjective findings such as tactile feedback during the perfusion process and direct visualization of adequate perfusion on fluoroscopy, which can be subject to user variability. Furthermore, it has yet to be determined how many freeze-thaw cycles the perfusate can undergo before image quality is compromised. In the current study, a perfused first metatarsal was freeze-thawed 4 times over the course of 2 months without a noticeable effect on the MicroCT image quality. This is important when considering multiple scans of a single specimen, such as before and after surgical intervention. Moreover, this perfusion technique is indicated for cadaveric research, and specimens required resection to fit within the small dimensions of a tabletop MicroCT scanner. Finally, as this is a cadaver-based model, anatomical fidelity and perfusate behavior may differ from in vivo conditions, limiting direct clinical translation.

In conclusion, this study presents a standardized and replicable MicroCT perfusion imaging technique for cadaveric evaluation of microvasculature in the foot and ankle. This model provides a foundation for future quantitative studies on microvascular integrity after surgical interventions, though its utility remains limited to cadaveric research.